Ion selective electrodes (ISE) operate on the measurement of the potential created when the equilibrium between an ion-pair is unbalanced, such as for example by the presence of an ion which disturbs the equilibrium or a substance such as an antibody which reacts with an antigen to create a potential which disturbs the equilibrium, between the ion-pair. An ISE includes a reference solution which comprises an aqueous solution of electrolyte and an ion-pair which is only slightly soluble in the aqueous solution. An ISE is useful for the measurement of concentrations of substances which cannot be detected by electrodes that depend upon the ionization of the substance being measured. Accordingly an ISE is used for the measurement of the concentration of organic compounds in a test solution or, when suitably modified in a manner known in the art, for the detection of bio-compounds.
The concept of biosensor was proposed more than twenty years ago. Numerous papers and patents describing various biological compounds associated to several sophisticated types of transducers for the assay of a variety of analytes, were issued. Since then, however, only a few biosensor based analyzers have become commercially available. It appears that the most commonly used biosensor is the enzyme electrode and the transducer is the amperometric probe for oxygen or hydrogen peroxide detection associated to an immobilized oxidase. The main biological sensitive elements used for design of biosensors are: enzymes, antibodies, organelles, bacteria or whole cells or tissue slices. The transducers generally used for design of biosensors are: amperometric or potentiometric electrodes, field effect transistors, piezoelectric crystals, thermistors, optoelectronic systems. The commercially available biosensor are used to analyze the glucose, enzymes and enzyme substrates. The typical example of described biosensor is disclosed in U.S. Pat. No. 4,894,253, issued Jan. 16, 1990.
The aromatic sulphonate ion-selective electrode (ISE) has been used in the prior art, for example, for the analysis of anionic detergents. As set forth in Talanta. 20, p. 867 (1973) a nitrobenzene solution of benzene sulphonate crystal violet was used as a liquid organic phase. The lower part of a glass U-tube (50 ml capacity) was filled with an organic phase of an ion-pair. Reference and sample solutions were separated from the organic phase and they were aqueous solutions of the same sulphonate as in the liquid organic phase. The reference and sample solutions were connected to saturated calomel electrodes through an agar bridge saturated with potassium chloride. The ISE had an approximately Nernstian slope (56 mV/log C) down to 1.times.10.sup.-4 M and was useful to 1.times.10.sup.-5 M. However the cited ISE has undesirable limitations, for example, the ISE is not sensitive and reproducible enough; the U-tube construction of the ISE body has open excess to nitrobenzene what is not convenient for analytical measurements and hazardous to use ecologically; the liquid organic phase exposure to solution of interest is not acceptable for biosensor development and hazardous ecologically.
I. A. Gur'ev at al. disclosed improvements of existing ISE technology in "Simplified the Design of a Liquid Ion Selective Electrode", Zavodskaja laboratoria, 46, No. 6, 497 (1980). This ISE was also used for the analysis of anionic detergents, its ISE organic phase consisted of a nitrobenzene solution of lauryl sulphate and crystal violet. The ISE was made as an adaptor to the silver/silver chloride electrode and the second silver/silver chloride electrode was used to complete the circuit. The lower part of the plastic tube was filled with fluoropolymer (USSR Trademark Floroplast F-4) that was swelled in the liquid organic phase before putting it in the tube and then compressed into the end of the tip of the adaptor tube. The liquid organic phase comprising a nitrobenzene solution of ion-pair lauryl sulphate crystal violet was placed above the polymer phase. A reference solution of 1.times.10.sup.-2 M potassium chloride was used. The ISE had a Nernstian slope (59 mV/log C) down to 1.times.10.sup.-6 M and was useful to 1.times.10.sup.-7 M. The ISE is more convenient to use when it is in an adapter form. The sensitivity of the ISE was increased ten times. The limitations of the described development are: the reproducibility was improved but not sufficiently to market this analytical method; the liquid organic phase leaks through the polymer composite phase during ISE operation; and the estimated life of the ISE is only about six months. A. Fisher disclosed certain aspects of the described ISE in an article entitled; "The use of ISE in Waste Water Analysis", The Method of Environmental Analysis, Moscow, 140-141, (1983).
Most applications of the ISE involved the analysis of detergents, nitrates, nitrophenols, sulfides and other compounds in ground and waste waters. Later A. Fisher published two articles: "Study of the Mechanism of the Functioning of a Liquid Membrane of an ISE Based on Ion-Pair Lauryl Sulphate Crystal Violet.", and "Asymmetry Potential of an ISE with a liquid Organic Phase Based on Ion-Pair of Lauryl Sulphate Crystal Violet.", Zhurnal Analyticheskoi Khimii., 40, No. 3, 5, 493-499, 935-936, 1985. The first article discusses the mechanism of the ISE functioning. In the second article it is disclosed that the ion-pair exists in the polymer composite phase in the form of liquid crystals.
Several articles are available on detergent electrodes with semipermeable phases and their use in the micelle cells. For example see: Kaibara K., et al. Mem. Fac. Sci. Kyushu Univ., Ser. C, 7, 1, (1970); Botre C., at al., Kolloid Z. Z. Polym., 250, 900, (1972); Birch B. J., at al., Anal. Chim. Acta, 69, 473, (1974); Senkyr J., J. Chem. Listy, 73, 1097, (1979). The ISEs constructed in accordance with the teachings of these articles have little selectivity and long response time. The last article describes an ISE with a liquid organic phase based on Crystal Violet which is used for nitrate analysis. This ISE has a concentration range 1.times.10.sup.-7 to 1.times.10.sup.-6 M and a surfactant potentiator factor of 10 to 2.5M. However, because the liquid organic phase leaks from the ISE, the useful life of the module is about 6 months.
Despite a considerable amount of work applied to the development of electroanalytical devices which can be used in a ISE or biosensor mode a need still exists for an electrode, and a simple and inexpensive method for making it, which exhibits reproducible response, rapid response time, selectivity and sensitivity which can be miniaturized. In addition there exists a need for a commercial device for the detection and measurement of biocompounds.